Previously we showed that the crystallins were recruited from stress proteins and enzymes and that some maintain dual roles without gene duplication. We have now begun an analysis of the promoter that specifies lens-preferred expression for the enzyme-crystallin, zeta-crystallin. We have continued to analyze the gene for alpah-enolase/tau-crystallin, which loses lens-preferred expression in cultured, lens-derived cells. The duck tau-crystallin gene has been introduced into transgenic mice to study expression in lens. The cDNA and gene for mu-crystallin, another enzyme-crystallin that achieves very high expression in some lenses, have been cloned. In a study on cold-shock domain DNA-binding proteins, we have cloned and sequenced mouse lens dbpB/YB-1, which is among the first DNA-binding proteins actually cloned from the lens. Others have suggested that dbpB/YB-1 is a negative regulator of MHC gene expression. We found that major histo-compatibility complex (MHC) gene expression is completely absent from normal lens. At the same time, we noticed that MHC class I genes have been activated in the transformed alphaTN4-1 cell line derived from mouse. Finally, we have discovered that proteins closely related, and perhaps identical to the lymphokines MIF and MRP8/cystic fibrosis antigen, are expressed in the lens. In the embryonic chick lens, 10K/MIF is developmentally regulated and may have a role in lens cell differentiation. We also discovered another lens-expressed protein that is related to a family of lipid- and retinoid-binding proteins, some of which are major substrates for the insulin receptor tyrosine kinase. Such a protein could mediate the effects of insulin on the lens. GRANTZ01EY00238 This project investigates the expression of proto-oncogenes during the differentiation of embryonic lens epithelial cells to form lens fiber cells and seeks to determine the specific function of the corresponding gene products in the developing lens. The normal developmental profiles of four nuclear proto-oncogene mRNA's (c-myc, N-myc, c-fos, and c-jun) have been determined by coupled reverse transcription and polymerase chain reaction. The results indicate that N-myc expression is associated primarily with the early stages of lens fiber cell differentiation in the annular pad and c-myc expression is strongly correlated with cell proliferation in the central epithelium and germinative zone, while c-fos and c-jun expression is highest in the quiescent cells of the central epithelium. Significant levels of c-myc mRNA have also been found in the annular pad cells, although no cell division occurs in this region. In situ hybridization using N-myc-specific and c-myc-specific riboprobes has confirmed that both mRNA's are expressed in the same population of annular pad cells. This finding suggests that the simultaneous expression of N-myc and c-myc may uncouple the usual association between c-myc and cell proliferation. Studies of DNA synthesis in the embryonic chicken lens have shown that the lens epithelium contains a population of cells that undergo DNA synthesis simultaneously. This synchronized cell population will be useful for in vivo analysis of cell cycle dependent gene expression.